The goal of this proposal is to determine the mechanisms underlying age-related changes in synaptic physiology at the neuromuscular junction. Acetylcholine (ACh) levels decrease with age. This manifests a change in the balance between synthesis and degradation. Thus, using sensitive gas chromatography/mass spectrometry, the rates of ACh synthesis and degradation will be measured to determine the nature of this age-related change. Intracellular Ca2+ levels may also be altered during aging. This will be tested using electrophysiological techniques. The kinetics of Ca2+ entry and clearance will be estimated from frequency histograms of synaptic delays. The relative amounts of Ca2+ entry will be measured using focal extracellular electrodes following pharmacological blocking of nerve-terminal K+ currents. The rate of clearance of intracellular Ca2+ will be estimated by measuring the time course for the decay of synaptic facilitation. Moreover, the sensitivity of the release mechanism to Ca2+ will be estimated from data relating quantal release to extracellular Ca2+ concentrations. Proteins weighing 125, 145, and 210 kDa are present in nerve terminals of 10-month but not 28-month rats. The 145 and 210 kDA molecules are quite probably neurofilament proteins. Using gel electrophoresis and immunoblot techniques, this identity will be tested. Similar techniques will be used to ascertain whether the failure to detect these proteins in aged rats is due to impaired axonal transport from the cell body or enhanced Ca2+-dependent degradation. In the postsynaptic membrane, there is an age-related increase in low-affinity, slow channel open time ACh receptors. Patch-clamp methods will be used to determine whether there are corresponding changes in mean channel conductance. Since these changes in receptor properties are similar to those following functional denervation, focal recording techniques will be used to test whether all nerve terminals are functional in the end plates of aged animals. These studies should elucidate the underlying causes of reduced ACh levels, altered synaptic efficacy, and changing end-plate structure during aging.